Transcript runt and engrailed-dependent specification of midline glia 1/25/10 Outline runt and engrailed specify AMG and PMG, respectively Runt and the midline The role of.

runt and engrailed-dependent specification of midline glia
1/25/10
Outline
runt and engrailed specify AMG and PMG, respectively
Runt and the midline
The role of hunchback and runt in the specification of MP1
Establishing early midline fate
Downstream targets of runt
Key developmental time points – AMG and PMG
Runt
En
AMG
PMG
AMG
AMG
PMG
PMG
AMG
AMG
MP1
MP1
Distinct expression patterns in AMG vs PMG
Runt and Engrailed are spatially restricted to either AMG or PMG
runt and engrailed control AMG and PMG gene expression, respectively
Wildtype
UAS-runt
UAS-en
Wildtype
UAS-runt
UAS-en
Previous work
runt expression
engrailed expression
WT
hs-Runt
Klinger and Gergen, 1993
Aronson, et al, 1997
Runt and Engrailed cross-repress each other in order to generate AMG and PMG
wildtype
When are expression domains established?
13 sag
Runt En GFP
13 sag
13 sag
Runt En
Runt
13 sag
Wrapper
Consequences of converting AMG to PMG or PMG to AMG
wildtype
late 16
GFP wrapper
UAS-runt
late 16
GFP
wrapper
UAS-en
late 16
GFP wrapper
?
L(1)sc
en
l(1)sc
runt?
Does engrailed regulate l(1)sc?
UAS-en
enE/enE
wildtype
L(1)sc Stg
runt negatively regulates l(1)sc
UAS-runt
y1 w1 f1 run3
L(1)sc Stg
L(1)sc Stg
12/1
L(1)sc Stg odd
L(1)sc
?
en
L(1)sc
runt
Remaining experiments
•Determine the spatial and temporal expression of Runt and Engrailed
•Determine role of L(1)sc
•Quantify glial cells in UAS-runt and UAS-en backgrounds
•Finish images for runt and engrailed mutant backgrounds
The specification of neuron and glial cell fate by the transcription factor Runt
1. Mechanisms used to generate MP1s
2. Establishment of early midline fate
3. Transcriptional regulation of AMG gene expression
Due April 8th (end of March for UNC?)
Runt
En
AMG
PMG
AMG
AMG
PMG
PMG
AMG
AMG
MP1
MP1
runt hb lim3
HGTX
AMG
odd
PMG
AMG
AMG
pdf
PMG
PMG
AMG
AMG
MP1
MP1
hb
AMG
PMG
?
PMG
PMG
?
?
MP1
MP1
?
Odd, HGTX, pdf, lim3?
sim::Gal4 driving uas-hb generates extra lim-3 HGTX + cells
WT
wt
lim3
WT control
(no HGTX staining)
stage 11 stage 13 stage 15 stage 16 stage 17
0
2
2
2
2
stg, uas-hb
lim3
UAS-hb
Lim3 GFP HGTX
Lim3 HGTX
stage 11 stage 13 stage 15 stage 16 stage 17
0.2
3 3.423077 3.043478 3.210526
Wrapper and lim3 co-localize in stg uas-hb embryos
Lim-3 + wrapper + neurons
Lim-3 + wrapper - neuron
lim3
stg
wrapper
Stop codon
deletion
Lim3 and odd are both expressed in hb12 alleles
lim3
stg
odd
runt hb lim3
HGTX
AMG
odd
PMG
AMG
AMG
pdf
PMG
PMG
AMG
AMG
MP1
MP1
Ectopic Runt expression does not affect Lim3 expression
WT stage 13
Stg uas-runt
GFP En Lim3
GFP En Lim3
WT
wt
lim3
stage 11 stage 13 stage 15 stage 16 stage 17
0
2
2
2
2
stg, uas-hb
lim3
stage 11 stage 13 stage 15 stage 16 stage 17
0.2
3 3.423077 3.043478 3.210526
stg, uas-runt
lim3
stage 11 stage 13 stage 15 stage 16 stage 17
0.00
2.04
2.09
2.00
2.00
Driving engrailed in the midline reduces odd expression
GFP odd Runt
GFP odd Runt
runt mutants lack odd staining
12/0
12/0
GFP odd
GFP odd
Hunchback and Runt?
HB
odd
HGTX
Runt
lim3
pdf
MP1 transcription factors
Lim3 ectopic expression?
Uas-odd
X
Uas-ftz
X
Uas-exex
X
exex-
Lim3 normal
Aim 1 Methods and goals:
•
Test available mutants and uas lines for altered MP1 fate
• Morphology
• gene expression
•
Identify MP1 enhancer
• Hunchback, Runt, and Engrailed binding sites
• Mutate sites and identify
•
Microarray MP1 neurons
• Isolate MP1s using glec enhancer and sim-GAL4 UAS-TAU-GFP (2-color sorting)
• Amplify and hybridize to affy microarray chip or use chip seq
1 kb
Runt binding site
Sim binding site
Hb binding site
Aim 2 – Early specification of midline by runt and additional transcription factors
eve
ftz
wingless
en
wingless
slp
wingless
patched
en
gooseberry-neuro – MP3s
Establishing early midline identity
A, B
l(1)sc
en, PMG
C
runt
MP1, hb, odd
D, E
runt, slp1 and 2
AMG
F
G
slp1 and 2, eve, en
gsb-n, l(1)sc
MP3
H
en
MNB, Vums
Aim 2: Establishing early midline identity
•
Test available mutants and uas lines for altered midline
• Morphology
• Gene expression
•Based on results, determine which transcription factors specify each cell type
Aim 3: Identify the transcriptional mechanisms used to generate AMG and PMG
Goals:
Identify Runt binding sites within AMG cis-regulatory regions
Identify Engrailed binding sites
Determine if Engrailed and Runt function in conjunction with Sim to
specify AMG and PMG cell fate
CG8776
1 kb
Sim binding site
Runt binding site
Summary
runt and engrailed co-repress each other to specify AMG and PMG fates, respectively
runt is necessary to generate MP1s, hunchback is sufficent
Further clues pertaining to midline development should arise
from uas lines and genetic analysis